While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above -80 °C without sample temperature alteration. Rat liver sections (1 cm2) were frozen at a rate of -1 °C/min to -20 °C, stored for 1 h at -20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to -80 °C methanol. After 1, 3, or 5 days of -80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at -20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at -20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.

中文翻译：

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高低温冷冻固定：一种观察组织内冰的技术

虽然目前存在用于观察储存在零以下高温度的组织内的冰的各种固定技术，但这些技术在评估不同的储存温度时需要不同的固定溶液组合物，或者改变样品温度以实现酒精-水替代。因此，开发了高低温冷冻固定 (HSC)，以促进在高于 -80 °C 的任何温度下进行固定，而不会改变样品温度。大鼠肝脏切片 (1 cm2) 以 -1 °C/min 至 -20 °C 的速率冷冻，在 -20 °C 下储存 1 小时，并使用经典冷冻替代 (FS) 或 HSC 进行处理。FS 样品浸入液氮中并保持 1 小时，然后转移到 -80 °C 的甲醇中。-80 °C 储存 1、3 或 5 天后，将样品置于干冰上的 3% 戊二醛中并使其升华。在转移到 4°C 之前，HSC 样品在 -20°C 的 HSC 固定剂中储存 1、3 或 5 天。在量化冰大小之前，将组织切片石蜡包埋、切片和染色。HSC 固定剂渗透与时间呈线性关系，可以通过数学建模来确定给定组织深度所需的固定持续时间。5 d 样品内部区域内的冰粒大小在 HSC 和 FS 处理之间是一致的（p = 0.76）；然而，FS 处理在组织的外部区域产生了更大的冰粒。这与 HSC 外部区域 (p = 0.016) 和 FS 内部区域 (p = 0.038) 显着不同。在 HSC 内部和外部区域之间没有观察到冰大小的差异 (p = 0.42)。这项工作表明，HSC 可用于观察储存在 -20 °C 的肝组织内形成的冰。